Optimized Expression and Highly Efficient Purification of the Anti-inflammatory Drug rIL-۱Ra from E. coli using Ni/Silica-Coated Magnetic Nanoparticles

Background: IL-۱β is a key mediator of inflammation in the body. Upon inflammasome activation, the IL-۱ receptor antagonist (IL-۱Ra) serves as the primary natural inhibitor of IL-۱β by competitively binding to its receptor, thereby limiting inflammatory signaling. Due to this mechanism, IL-۱Ra has garnered significant interest as a biological anti-inflammatory drug.Objectives: This study aimed to produce recombinant IL-۱Ra (rIL-۱Ra) in E. coli using optimized expression conditions and to develop a highly efficient purification process utilizing Ni/silica-coated magnetic nanoparticles.Methods: The IL-۱Ra gene was cloned into the pET-۲۸a expression vector. The correct construction of the recombinant plasmid was verified by PCR and DNA sequencing. Expression of rIL-۱Ra was carried out in E. coli BL۲۱ (T۷ Express) under optimized conditions (induction with ۰.۵ mM IPTG at ۲۵ °C for ۱۶-۱۸ h). The expressed protein was analyzed by SDS-PAGE and Western blot. Purification was performed using Ni/silica-coated magnetic nanoparticles, followed by protein concentration via polyethylene glycol (PEG). The protein concentration was determined by Bradford assay, and the product was subsequently stabilized by buffer exchange into PBS (pH ۷.۴) through dialysis, supplemented with ۱۰% glycerol, and stored at -۲۰ °C.Results: PCR and sequencing confirmed the successful construction of the expression cassette, showing the expected ~۴۵۰ bp insert. SDS-PAGE and Western blot analyses detected a protein of approximately ۱۹.۸ kDa, confirming the expression and identity of rIL-۱Ra. Maximum soluble expression was achieved under the optimized conditions. Purification using Ni/silica-coated magnetic nanoparticles yielded ۱۰ mg of rIL-۱Ra per ۱۰۰۰ ml of bacterial culture (۱۰ mg/L).Conclusion: The E. coli BL۲۱ (T۷ Express) system proved to be an effective and cost-efficient host for producing soluble rIL-۱Ra. Furthermore, the use of Ni/silica-coated magnetic nanoparticles provided an efficient and scalable purification method, yielding a substantial amount of the recombinant protein suitable for further research and potential therapeutic applications.

کلیدواژه ها:

Interleukin ۱ Receptor Antagonist Protein ، arthritis ، Rheumatoid ، anti-inflammatory agents ، Magnetite nanoparticles